Process for the enzymic unhairing of hides

ABSTRACT

A PROCESS FOR THE UNHAIRING OF HIDES COMPRISING A PRETREATMENT BY SOAKING THE HIDES IN A SOLUTION OF ALKALI METAL HYDROXIDE FOLLOWED BY THE APPLICATION OF A PROTEOLYTIC ENZYME MATERIAL CONTAINING CHYMOTRPTIC ACTIVITY, AS DETERMINED BY ASSAY ON ACETYL TYROSINE ETHYL ESTER, IN AN AMOUNT SUFFICIENT TO PROVIDE FROM ABOUT 0.0005% TO 0.05% CHYMOTRYPTIC ACTIVITY BASED ON THE WEIGHT OF THE HIDES.

United States Patent Ser. No. 82,154

Int. Cl. C12b 1/00 US. Cl. 195-6 8 Claims ABSTRACT OF THE DISCLOSURE A process for the unhairing of hides comprising a pretreatment by soaking the hides in a solution of alkali metal hydroxide followed by the application of a proteolytic enzyme material containing chymotryptic activity, as determined by assay on acetyl tyrosine ethyl ester, in an amount sufficient to provide from about 0.0005% to 0.05% chymotryptic activity based on the weight of the hides.

COPENDING APPLICATION This application is a continuation-in-part of our application Ser. No. 666,958 filed Sept. 11, 1967, now abandoned.

BACKGROUND OF THE INVENTION (1) Field of invention The invention of the present application, as stated above, is directed to a process for the removal of hair from hides (and skins) to prepare them for tanning.

(2) Description of the prior art The most widely practiced systems of commercial unhairing involve the exposure of hides to a suspension of hydrated lime (liming), usually with the addition of activators, such as sodium sulphide or other active sulphur, agents, dimethylamine, and/or other reducing agents, to the liming bath. After dehairing, the skins must be subjected to deliming, bating and/or other treatment prior to tanning.

Several problems are inherent in the existing commercial systems, namely:

(a) Degradation of the leather-making protein, collagen, occurs during prolonged exposure to conditions of high alkalinity, which, in fact, is one of the methods used for the conversion of collagen into gelatin. Such treatment, therefore, results in leathers of lowered physical strengths.

(b) Hair, which is a valuable by-product, is weakened by the action of reducing agents (such as sulphides) and by extremes of alkalinity.

(c) The cystine of hair is degraded. Untreated cattle hair may contain 10-11% cystine, while by-product hair from a commercial tannery contains as little as 2% cystine, which makes it unsuitable as a source of this amino acid.

(d) The agents, especially sulphides, used to loosen the hair are both unpleasant to handle and toxic. Used depilatories pose serious disposal problems.

The use of enzymes in unhairing and bating operations is well known in the art. For example, US. Pat. No. 1,082,911, granted to Rohm in 1913, describes a process for unhairing utilizing an alkaline solution containing a tryptic enzyme. This system has been used satisfactorily for some leathers, but has not been found suitable for cattle hides. Numerous systems have been suggested in recent years, using enzymes derived from microorga- 3,679,548 Patented July 25, 1972 nisms, for example, B. s'ubtilis, A. oryzae, A. parasiticus, P. vulgaris, S. fradiae, S. albus G, etc., from plants such as pineapple and papaya and from mammalian tissues.

Despite the numerous enzymic systems suggested for depilation, none has gained general acceptance. Some require the use of such large quantities of enzymic agents as to be quite uneconomical. (Note, for example, J. Am. Leather Chemists Assn., 1961, 56, page 31, and US. Pat. Nos. 2,095,273, 3,133,002 and 3,203,868.)

In some instances, the sources of the enzymes are so poorly defined that it is not possible to consistently reproduce a workable enzyme system, e.g. definitions such as fungal enzymes, bacterial enzymes, proteases, and saccharases cover a wide variety of enzyme products and provide no guidance for preparing a practical reproducible unhairing system. Moreover, the leathers resulting from known enzyme unhairing systems exhibit a variety of deficiencies, among which can be mentioned low yields, incomplete removal of hair, drawn grain, firmness and loose grain.

There has been no body of experimental evidence showing the mechanism of enzymic unhairing. As a result, disagreements have arisen as to the nature of the enzymes required for the purpose. Thus, depilatory activity has been variously attributed to proteases, amylases, elastases and mucases. The only specific enzyme which has been named repeatedly in the literature is the protease, trypsin. In addition, the literature contains no reliable method for the assay of depilatory activity, and although many assay systems have been suggested, none has been found to correlate satisfactorily with depilatory activity.

BRIEF SUMMARY OF THE INVENTION The present invention has for its object the avoidance of the problems of the prior art and the provision, for the first time, of a satisfactory reproducible enzymic unhairing system. We have discovered that the chymotryptic activity of a proteolytic enzymic material provides the key for determining the proper level of enzymic material to be applied during a hide unhairing process.

The invention comprises the combination of a short presoak in alkali metal hydroxide solution, followed by application of a proteolytic enzyme material containing chymotryptic activity as determined by assay on acetyl tyrosine ethyl ester in an amount sufficient to provide from about 0.0005% to 0.05% by weight, based on the weight of the hides, of chymotryptic activity as so determined. The treatment is mild (bot'h chemically and mechanically) in its action on hides. With enzyme material containing chymotryptic activity the alkali metal hydroxide pretreatment of the hides greatly increases the elfectiveness of the depilatory system. Such enzyme material has no significant depilatory activity on intact hidesthat is, on hides which have not undergone alkali pretreatment.

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DETAILED DESCRIPTION According to the invention, the alkali pretreatment is achieved by soaking the hides, which may be either fresh, cured or frozen, for a period of about 12-24 hours, e.g., overnight, at a temperature below about 30 C. with 0.015 to 0.360 mole per liter of alkali metal hydroxide. agitation in a medium which, initially, contains about The pH is initially in the range of about 11 to 13 but drops during the soaking period. At higher levels of alkali, a subsequent buflering may be used to bring down the pH. The soaked hides are then treated with a chymotrypsin enzyme preparation in suflicient quantity to provide from about 0.0005 to,0.05% chymotryptic activity based on the weight of the hide. The enzyme is conveniently applied in aqueous solution or suspension in the pH range 6-10. For control of bacterial populations it may be desirable to keep the pH of the enzyme me dium as high as possible consistent with good hair removal. A pH of between about 8.5 and 9.5 is preferred for this purpose. The enzyme preparation may be'applied in a conventional tanning drum or other convenient manner. The enzyme-treated hides are then stored at about 18-50 (3., preferably 35-43 C., for about 2072 hours. The hair is then washed off or removed by other gentle action.

Various sources of the proteolytic enzyme may be used, provided the necessary level of chymotryptic activity is present in the system. Thus, commercially available chymotrypsin in crystalline form may be employed, as well as such materials as pancreatin, activated pancreatic wastes, and any other material which upon assay on acetyl tyrosine ethyl ester as a substrate is found to contain a practical amount of chymotryptic activity. chymotryptic activity may vary widely from batch to batch of proteolytic enzyme material. For example, in pancreatin material from beef sources the chymotryptic activity of one batch maybe almost double that of another batch. The chymotryptic activity of pancreatin from beef sources is also much higher, in general, than that from hog sources. In order to control the unhairing process with the proteolytic enzyme material it is essential to know the chymotryptic activity of the batch being used. It will be understood that materials low in chymotryptic activity, as determined by quantities to provide the necessary level of 0.0005 to 0.05% chymotryptic activity.

Since there is little agitation during the process, defects resulting from excessive mechanical action do not occur with this process. Hair removal is substantially complete, and the resulting hair is a by-product of greater value than that obtained from liming processes, since there has been little degradation in physical strength or in cystine content. Waste disposal is not a problem, since the spent .depilatory liquor is of negligible volume and contains only pollutants which are readily oxidized biologically.- The depilatory agent is not toxic, and the hazard associated with the use of caustic soda is not as great as that associated with the sulphides used in current practice.

Assay methods.

. The force required to pull an individual hair from its follicle has been called the depilation load (D.L.) The assay of depilatory activity is performed routinely by comparing averagedepilation loads before and after enzymic treatments. The change in depilation load is customarily expressed as a percentage (A D.L. percent) of the average value before treatment. Each such figure is normally the average of 60 values, obtained from six different hides,

with ten values per hide.

It has been found that the depilation load must be reduced to an average absolute level of about 3 grams for practical unhairing with commercial unhairing machines. A similar level seems adequate for removal by washing. This corresponds to a value for A D.L. percent or at least about -90%.

The assay of enzyme material for chymotryptic activity is performed on a Radiometer pH-Stat (manufactured by the Radiometer Company, Copenhagen NV, Den- .ma-rk). The synthetic substrate acetyl tyrosine ethyl ester l the aforesaid assay, will have to be used in relatively large 1 Micromoles of acid produced per minute per mg. of enzyme.

1 Available as a waste product from the commercial extraction of zymo- I gens by the methodof Kunitz and Northrop, J-'G8H.'Ph YSlOL, 1035, 18, 433, and activated to its maximum activity using pancreatm as activator- Having thus generally described the invention, specific operative embodiments will be set forth with the understanding that such embodiments are not to be construed as restrictive of the invention as a whole.

EXAMPLE I Sixty-gram samples of bovine hides were soaked overnight in 0.03 M sodium hydroxide. Each lot was then buffered using a phosphate buffer (pH 7.0, ionic strength 1.2) and each medium contained 2% (v./v.) chloroform to minimize bacterial growth. Treatment was then carried out using enzymes, as set forth below, applied to the hair surfaces, and storing for 24 hours at 37 C.

The results determined were as follows:

Quantity of enzyme (percent A D.L. Enzyme used w./w. (percent) None (control) 13 Pancreatin 0. 02 Chymotrypsin. 0. 0015 -100 Trypsin. 0. 0017 -5 Elastase 0. 00023 -11 l The amounts of trypsin, ehymotrypsin and elastase were selected to correspond approximately with the amounts found in 0.04% pancreatin.

EXAMPLE II Calf-, sheepand pigskin samples were soaked as in Example I. The skins were then buffered in phosphate buffer having a pH of 6.5 and an ionic strength of 1.2 and painted with a suspension of pancreatin (0.02% w./w.) in the same buffer. Storage was at 37 C. The following lots were completely unhairedz' (a) fresh calfskins, treated 48 hours.

(b) fresh-frozen calfskins, treated 24 hours. (c) dry-salted calfskins, treated 48 hours. (d) fresh sheepskins, treated 48 hours.

(e) dried sheepskins, treated 48 hours.

(f) fresh pigskins, treated 24 hours.

EXAMPLE III Samples of bovine hides were obtained fresh. After demanuring and defleshing, they were washed thoroughly in cold water and treated with various concentrations and volumes of sodium hydroxide. Each lot was then buffered with phosphate buffer (pH 6.3; ionic strength 1.2) and treated on the hair side with a suspension of 0.02% pancreatin in the buffer. The changes in depilation loads were determined over 20-hour intervals at 37 C. The results were as follows:

Eflect of Sodium hydroxide treatment enzyme treatment, Volumezhide A D.L.

Concentration ratio (percent) EXAMPLE Iv Samples of fresh steerhides were soaked overnight in 0.03 M sodium hydroxide. They were then equilibrated with phosphate buffer (pH 6.3; ionic strength 1.2) and treated on the hair side suspensions of 0.01% (w./w. pancreatin 3.6 x N.F. in the same buffer. After 20 and 48 hours of treatment at a variety of temperatures, the results were as follows:

A D.L. (percent) It was found that a certain batch of hides (available at the time of this test) would pick up 14:2.4% moisture when dipped in water for 15 seconds. The minimum pickup, therefore, was about (l42 2.4=) 9% moisture. Sufiicient enzyme was dissolved in water so that hides picking up the minimum amount of moisture would also pick up enough enzyme for unhairing.

Thus, samples of brine-cured steerhides were washed 30 minutes, drained and treated for 24 hours with 400% (v./w.) sodium hydroxide solution (0.03 M). They were then drained, fleshed and dipped in pancreatin (0.01 g./m1.) for 15 seconds. After storage for 24 hours at 37 C., the hair on all samples was removed easily.

EXAMPLE VI Samples of brine-cured steerhide (3" x 3") were washed one hour in running tap water, then soaked 20 hours in 400% (v./w.) of sodium hydroxide (0.03 M). Some were then dipped in a suspension of pancreatic residue such that 0.3% (w./w.) of the solid residue was applied. Control samples were dipped in suspensions of pancreatin such that 0.02% (w./w.) was applied. All samples were incubated at 37 C. for 20 hours in contact with chloroform vapour. Unhairing was satisfactory with both treatments.

EXAMPLE VH Fresh samples of steerhide (15 g. lots) were soaked overnight in 0.03 M solutions of (a) lithium hydroxide, (b) potassium hydroxide, (c) barium hydroxide, and (d) lutidines. Each lot was then bufli'ered with phosphate (pH 7.0, ionic strength 0.15) and painted with 0.1% (w./w.) pancreatin in aqueous suspension. After incubation at 37 C. for six hours, the hair was loosest on (a) and tightest on (d); after 72 hours all four lots unhaired easily and completely.

EXAMPLE VIII Samples of hide (six pieces in each lot) were prepared as in Example I, with the exceptions that the base used in the soak was varied (as tabulated below), and the buffer used prior to enzymic treatment was of ionic strength 0.2 (phosphate, pH 7.0). All the pieces were painted with 0.01% pancreatin 3.6 x NR and incubated at 37 C. for 24 hours. The assay of depilase activity over this interval was made in the previously described manner, with the results shown in Table I.

TABLE I Effectiveness of enzyme treatment, Concentration A D.L. Group Lot Base used of base (percent) 1 Sodium hydroxide 30 mM 1 93 I 2 Ammonium hydroxide 20mM -56 3 30 mM -78 4 15 mM 54 5 Sodium hydroxide 30 mM -94 2 Calcium hydroxide- Saturated (about 47 25 mM II 7 do 0.6 Saturated 74 (about 12 mM). 8 do 0.25 Saturated 69 (about 6 mM). 9 Sodium hydroxide 30 mM 92 III- 10 Dimethylamine 60mM 68 11 30 mM 64 12 do 15 mM 66 l 1 mM=0.001 molar.

EXAMPLE 1X Samples of steer hides were washed and then soaked with various concentrations of sodium hydroxide ranging from 0.12 to 0.36 molarity, in a volume ratio of 4 to 1 sodium hydroxide solution with respect to the hides, at a temperature of 25 C. for 24 hours. The samples were then buffered at approximately pH 8.5 to 9.5 by use of a buffer solution containing ammonium sulfate as the buffer salt. The buffered hides were then incubated at 37 C. for 24 hours in vol./wt. of hides of pancreatin solution containing 0.2% pancreatin (0.009% chymotryptic activity based on the weight of the hides). Satisfactory unhairing was obtained in each instance by washing the treated hides in a stream of water. The recovered hair was of good quality except for the highest concentration of sodium hydroxide. The tests are summarized as follows:

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The entire operation as described above can be conducted on whole hides in a conventional tanning drum with, for example, an initial thirty minute wash cycle in water during which the drum is revolved, a 24 hour alkali metal hydroxide solution soak with periodic or slow revolution of the drum, a fifteen minute buffer soak in buffer solution with revolution of the drum, a 24 hour enzyme soak with enzyme solution supplying chymotryptic activity in the range of 0.0005 to 0.05% based on the weight of the hides with periodic or slow revolution of the drum and a final water wash of about 10 minutes with revolution of the drum to remove the hair. To preserve hair quality the alkali metal hydroxide solution is preferably below about 0.36 molarity.

SUMMARY The method of the present invention for unhairing of hides comprises the novel combination of the following features:

(1) Prior to enzymic treatment, the hides should be treated with alkali metal hydroxide solution of at least 0.015 molarity.

(2) Hides pretreated with alkali metal hydroxide sol-ution should be exposed to enzymes, having chymotrypsin activity as shown by assay on acetyl tyrosine ethyl ester.

(3) Application of the chymotrypsin enzyme should be with solutions or suspensions of amounts such that the hide is exposed to chymotrypsin enzyme equivalent in activity to 0.0005% to 0.5% chymotrypsin based on the weight of the hide.

It is to be understood that various modifications will readily become apparent to those skilled in the art upon reading the foregoing description of the invention. All

such modifications are intended to be included within the scope of the invention as defined in the appended claims.

' We claim:

1; In a method of dehairing hides comprising the treatment of the hides with proteolytic enzymic material, the improvement comprising: pre-soaking said hides in alkali metal hydroxide solution, determining the level of chymotryptic activity of said proteolytic enzymic material by assay on acetyl tyrosine ethyl ester and then treating said alkali pre-soaked hides with an amount of said enzymic material sufficient to provide from about 0.005% to 0.05% by weight, based on the weight of the hides, of

chymotryptic activity as previously determined.

' 2. The method of claim 1 wherein said alkali metal hydroxide solution is a solution of sodium hydroxide and wherein said proteolytic enzymic material is pancreatin.

' "3. A method for dehairing" hides comprising: 'soakin'g having a the hides in alkali metal hydroxide solution initially having a molarity of from about 0.030 to 0.360 for a period of about 12 to 24 hours; applying aqueous enzymic material to the pre-soaked hides, said enzymic material containing an enzyme having chymotryptic activity as determined by assay on acetyl tyrosine ethyl ester "and being selected from the group consisting of com-- mercial chymotrypsin, pancreatin, and pancreatic waste residues, the amount of enzymic material applied being suflicient to provide chymotryptic activity equivalent to from about 0.005% to 0.05% by. weight of crystalline chymotrypsin based on the weight of the hides; storing the enzyme treated hides at about 18 to C. for up to 72 hours; and removing the hair from the stored hid'es.

4. The method of claim 3 wherein the alkali metal hydroxide solution is sodium hydroxide solution.

5. The method of claim 3 wheerin the hides after alkali pretreatment are treated with aqueous enzymic material I at a pH of about 8.5 to 9:5.

6. The method of claimd wherein theenzymic material comprises commercial chymotrypsin.

7. The method of claim 3 wherein the enzymic material comprises pancreatin;

8. The method of claim Swherein the enzymic material comprises pancreatic waste residues.

., N s sr sss C ed:

Roddy et al., J. Am. Leather Chem. Assoc, vol. LVII, pp. 624-37 (1963).

A. LOUIS MONACELL, Primary Examiner G. M. NATH, Assistant Examiner UNITED STATES PATENT OFFI E CERTIFICATE OF CORRECTION Patent No. 3 I 7 548 Dated July 25 1972 ROSS GRANT DONOVAN and STEWART FRANKLIN WOLF Inventor(s) It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 2,- lines 63 and 64 have been reversed and should read as follows --a.gitation in a medium which, initially,

contains about 0.015 to 0.360mole-per liter of alkali metal hydroxide.

Signed and sealed this 25th day of December 1973.

(SEAL) Attest:

EDWARD M.FLETQHER,JR. RENE D. TEGTMEYER Attesting Officer Acting Commissioner of Patent:

FORM PO-IOSO (10-69) v USCOMWDC 6o376 p91 'h u.s. e'ovanunsm- PRINTING'OFFICE: lacs o--:sss-a:u' 

